The human metapneumovirus (hMPV) is an important viral agent associated with severe infections of the upper and lower airways, especially in young children and immunosuppressed subjects. Nevertheless, in vitro studies of hMPV are very difficult due to the little knowledge we have on its laboratory manipulation. OBJECTIVE The aim of this study was to isolate and propagate hMPV from patients, and to establish a method to quantify the virus by plaque assay. METHOD As part of a Latin American respiratory virus surveillance study, 12 nasal secretion samples - hMPV-positive by direct fluorescence - were inoculated on LLC-MK2 cells to isolate the virus. The supernatants were re-inoculated and the cytopathic effect and syncytium formation were evaluated daily; the infection was confirmed by immunofluorescence and RT-PCR. A protocol to titrate the harvested virus was established inoculating serial dilutions on LLC-MK2 cells, and agarose was then added as an overlay. After different time periods, the monolayers were fixed and stained with Naphthol blue/black or crystal violet and finally the viral titer was obtained. RESULTS Eight out of 12 hMPV-positive respiratory samples were positive for the isolation and confirmed by RT-PCR and immunofluorescence, but the cytopathic effect and syncytium formation were observed only in 5 cultures. One out of 8 viral isolates was used for propagation and plaque assay standardization. We found that incubation for 7 days in the semisolid overlay yielded plaques with appropriate size and shape to be counted, although crystal violet staining showed slightly larger plaques than those seen with Naphthol blue/black staining. CONCLUSIONS The isolation and propagation from patient-derived hMPV and the standardization of a practical, reliable, and inexpensive method of detection and quantification of hMPV were carried out, without the additional use of antibodies that had not been reported previously. These results offer some important insights for future studies of cellular and molecular biology of hMPV.

中文翻译：

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人间质肺炎病毒：分离，繁殖和噬斑滴定的实验室方法。

人间质肺炎病毒（hMPV）是与上呼吸道和下呼吸道的严重感染相关的重要病毒剂，尤其是在幼儿和免疫抑制的受试者中。然而，由于对hMPV的实验室操作了解甚少，因此其体外研究非常困难。目的本研究的目的是从患者中分离和繁殖hMPV，并建立一种通过噬斑测定法对病毒进行定量的方法。方法作为拉丁美洲呼吸道病毒监视研究的一部分，在LLC-MK2细胞上接种了12个鼻分泌物样品-通过直接荧光检测hMPV阳性，以分离出该病毒。再次接种上清液，并每天评估其细胞病变作用和合胞体形成。通过免疫荧光和RT-PCR证实感染。建立了滴定收获病毒的方案，将连续稀释液接种到LLC-MK2细胞上，然后添加琼脂糖作为覆盖物。在不同的时间段后，将单层固定并用萘酚蓝/黑色或结晶紫染色，最后获得病毒滴度。结果12份hMPV阳性呼吸道样本中有8份为分离阳性，并通过RT-PCR和免疫荧光证实，但仅在5份培养物中观察到了细胞病变作用和合胞体形成。8个病毒分离株中有1个用于繁殖和噬菌斑测定标准化。我们发现，在半固体覆盖层中孵育7天会产生具有适当大小和形状的菌斑，尽管结晶紫染色显示的菌斑比萘酚蓝/黑染色的菌斑稍大。结论进行了从患者来源的hMPV的分离和繁殖，以及一种实用，可靠，廉价的hMPV检测和定量方法的标准化，无需额外使用以前未报道的抗体。这些结果为hMPV的细胞和分子生物学的未来研究提供了一些重要的见识。